A. Hyperexpression and Purification of General Proteins of the Escherichia coli Phosphoenolpyruvate:sugar Phosphotransferase System. Genes for the proteins known as Enzyme I, HPr and Enzyme III (specific for glucose) were cloned into an expression vector to make three unique constructs. The proteins of interest were expressed to high levels. Simple purification procedures to prepare essentially homogeneous preparations of the three proteins were devised. B. Analysis of Deletion Mutations in the Gene for Escherichia coli Adenylylcyclase. Two spontaneous deletions in the E. coli adenylylcyclase gene (cya) devoid of catalytic activity were characterized. Sequence analysis indicated that one of them resulted in a 25 amino acid in frame deletion in the amino-terminal region of the protein while the other was a 41 base pair deletion that resulted in a frame shift with premature translation termination. Both mutations were in the same region of the gene and were characterized by unique sequence repeats at the termini of the deletions. C. Site-Specific Mutagenesis of a Presumptive ATP Binding Site in Escherichia coli Adenylylcylase. The sequence of seven adenylylcyclases were examined for the presence of domains typical of ATP binding sequences. A unique consensus that differed from that previously described for other types of ATP binding proteins was identified. The conserved amino acids of E. coli adenylylcyclase were modified by oligonucleotide-directed mutagenesis. The mutated forms of the enzyme were examined by photoaffinity labelling with 8-azido-ATP as well as by kinetic studies of catalytic activity. The results support the identification of the consensus region as an important ATP binding domain of the E. coli enzyme.